Most human cancers evolve over a period of years, starting as hyperplastic non-invasive lesions and progressing to highly invasive and metastatic malignant tumors. This process is in most cases associated with a gradual increase in genomic instability, such that hyperplastic lesions have very few genetic loci with loss-of-heterozygosity (LOH), while in advanced cancers LOH involves a large fraction of genetic loci. In parallel, tumor progression is associated with mutations in key growth controlling genes, such as p53. The high frequency of p53 mutations in human cancer raises the possibility that human cancer might be associated with deregulation of the DNA double-strand break (DSB) checkpoint pathway, because p53 is a component of this pathway. To better understand the natural history of cancer development in humans we examined various DNA damage response markers in a spectrum of lung lesions ranging from hyperplasia to invasive carcinoma. Unexpectedly, in the hyperplastic lesions there was evidence of a DNA damage response, as indicated by H2AX and Chk2 phosphorylation, 53BP1 focal staining, p53 accumulation and a high frequency of apoptosis. Progression to dysplasia and carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. Further, human skin xenografts induced to become hyperplastic by growth factors were characterized by H2AX and Chk2 phosphorylation and p53 accumulation and apoptosis. Based on this analysis we hypothesize that, even in its earliest stages, cancer development is associated with formation of DNA DSBs. Because the DNA DSBs form so early in cancer development, we also hypothesize that they are not linked to telomere attrition, but rather to aberrant DNA replication. We further hypothesize that the presence of DNA DSBs provides the selective pressure for p53 mutations in cancer, as well as the driving force for genomic instability. In this application we propose experiments to test our hypothesis. We believe that the proposed studies can change the way we view human cancer development and progression and provide the potential for novel cancer therapies aiming to exploit the presence of DNA DSBs in cancer, but not normal, cells.